Methods :
Primary Human Microvascular Endothelial Cells (HRECs), from the same donor, were cultured using endothelial-specific media and techniques. Two isoforms of VEGFA-165 were utilized, one with an interaction site for the Neuropilin-1 coreceptor, and a b-isoform of VEGFA-165 that lacks the Neuropilin-1 interaction sequence. Dose responses were assayed using infra-red (IR) immunofluorescent labeling of activated MAPK, AKT and VEGFR2. For normalization, beta-Actin was co-labeled with a different IR tag. Dose response curves were fit using the DRC-package in the R-project statistical environment within OS-X.

Results :
Activation of MAPK & AKT displayed a sharp pseudo-binary response to regular VEGFA-165 that contained the Neuropilin-1 interaction site. In contrast, activation responses were more gradual and less sensitive using an isoform of VEGFA-165 that lacked the Neuropilin-1 interaction sequence. ED50 values were 300-800 pM greater than for regular VEGFA-165. VEGFR2 activation displayed a similar large relative shift to sharper response.

Conclusions :
The C-terminus of VEGFA-165, which interacts with Neuropilin-1, dramatically decreases the ED50 values for the activation of both MAPK & AKT, creating a pseudo-binary response. This phenomenon was also seen for activation of VEGFR2. Our results reveal that it may be very difficult to titrate activation levels using the current VEGF blockade and traps. However, there may be room to reduce the high doses of anti-VEGF currently in use, now that we have determined the first dose response curves for Human Retinal Endothelial Cells.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.